In vivo transduction with a chimeric aav capsid protein

ABSTRACT

Recombinant adeno-associated viral (AAV) capsid proteins are provided. Methods for generating a library of recombinant adeno-associated viral capsid proteins are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of US application Ser. No. 13/472,260,filed May 15, 2012, now allowed, which is a continuation of USapplication Ser. No. 13/297,110, filed Nov. 15, 2011, now U.S. Pat. No.8,574,583, which is a divisional application of US application Ser. No.12/538,791 filed Aug. 10, 2009, now U.S. Pat. No. 8,067,014, which is acontinuation of US application Ser. No. 11/731,314 filed on Mar. 30,2007, now U.S. Pat. No. 7,588,772, which claims priority to U.S.Provisional Application Ser. No. 60/787,371, filed on Mar. 30, 2006.Each of the aforementioned applications and patents is incorporatedherein by reference in its entirety.

STATEMENT REGARDING GOVERNMENT INTEREST

This invention was made with Government support under contracts HL064274and HL066948 awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

A “Sequence Listing” is submitted with this application in the form of atext file, created Nov. 7, 2014, and named “586008243US04seqlist.txt”(28048 bytes), the contents of which are incorporated herein byreference in their entirety.

TECHNICAL FIELD

The subject matter described herein relates to libraries of recombinantadeno-associated viral (AAV) plasmids or viruses with varying capsidnucleotide sequences and to methods of generating the libraries. Thesubject matter also relates to nucleotide sequences isolated from thelibraries and to the AAV capsid proteins encoded by these sequences. Thesubject matter also relates to plasmids and viruses comprising theidentified sequences, which preferably provide a high transductionefficiency and a low level of neutralization by the human immune system.

BACKGROUND

Multiple recombinant gene transfer vectors based on different types ofviruses have been developed and tested in clinical trials in recentyears. Gene transfer vectors based on adeno-associated virus (AAV),i.e., AAV vectors, have become favored vectors because ofcharacteristics such as an ability to transduce different types ofdividing and non-dividing cells of different tissues and the ability toestablish stable, long-term transgene expression. While vectors based onother viruses, such as adenoviruses and retroviruses may posses certaindesirable characteristics, the use of other vectors has been associatedwith toxicity or some human diseases. These side effects have not beendetected with gene transfer vectors based on AAV (Manno et al., NatureMedicine, 12(3):342 (2006)). Additionally, the technology to produce andpurify AAV vectors without undue effort has been developed.

At least 11 AAV serotypes have been identified, cloned, sequenced, andconverted into vectors, and at least 100 new AAV variants have beenisolated from non-primates, primates and humans. However, the majorityof preclinical data to date that involves AAV vectors has been generatedwith vectors that are based on the human AAV-2 serotype, which isconsidered the AAV prototype.

There are several disadvantages to the currently used AAV-2 vectors. Forexample, a number of clinically relevant cell types and tissues are notefficiently transduced with these vectors. Also, a large percentage ofthe human population is immune to AAV-2 due to prior exposure towildtype AAV-2 virus. It has been estimated that up to 96% of all humansare seropositive for AAV-2, and up to 67% of the seropositiveindividuals carry neutralizing anti-AAV-2 antibodies which couldeliminate or greatly reduce transduction by AAV-2 vectors. Moreover.AAV-2 has been reported to cause a cell mediated immune response inpatients when given systemically (Manno et al., Nature Medicine,12(3):342 (2006)).

Methods of overcoming the limitations of AAV-2 vectors have beenproposed. For example, randomly mutagenizing the nucleotide sequenceencoding the AAV-2 capsid by error-prone PCR has been proposed as amethod of generating AAV-2 mutants that are able to escape theneutralizing antibodies that affect wildtype AAV-2. However, it isexpected that it will be difficult to generate significantly improvedAAV-2 variants with single random point mutations, as the naturallyoccurring serotypes have only about 85% homology at the most in thecapsid nucleotide sequence.

Methods of using a mixture of AAV serotype constructs for AAV vectorshave also been developed. The resulting chimeric vectors possess capsidproteins from different serotypes, and ideally, thus have properties ofthe different serotypes used. However, the ratio of the different capsidproteins is different from vector to vector and cannot be consistentlycontrolled or reproduced (due to lack of genetic templates), which isunacceptable for clinical use and not satisfactory for experimental use.

A third approach at modifying the AAV-2 capsid are peptide insertionlibraries, in which randomized oligonucleotides encoding up to 7 aminoacids are incorporated into a defined location within the AAV-2 capsid.The display of these peptides on the AAV-2 capsid surface can then beexploited to re-target the particles to cells or tissues that areotherwise refractory to infection with the wildtype AAV-2 virus.However, because knowledge of the atomic capsid structure is aprerequisite for this type of AAV modification, this method is currentlyrestricted to AAV serotype 2. Moreover, peptide insertion librariestypically cannot address the issues of AAV particle immunogenicity ortransduction efficiency.

Thus, there remains a need for new AAV vectors and a method ofgenerating new AAV vectors. In particular, there is a need for AAV basedvectors that can be used efficiently with a variety of cell types andtissues and that do not react with a pre-existing anti-AAV humanimmunity that could neutralize or inactivate the vectors. There alsoremains a need for vectors that transduce different cell types in vivoand in vitro and that offer a more restricted biodistribution or a morepromiscuous biodistribution, depending on what may be required. Inparticular, there remains a need for vectors capable of transducing avariety of cells types, such as hematopoietic stem cells or embryonicstem cells.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification and a study of the drawings.

BRIEF SUMMARY

The following aspects and embodiments thereof described and illustratedbelow are meant to be exemplary and illustrative, not limiting in scope.

In one aspect, recombinant capsid proteins and methods for generatingrecombinant capsid proteins are provided. The capsid proteins includeregions or domains that are derived from different serotypes of AAV. TheAAV serotypes may be human or non-human. Recombinant AAV comprising thecapsid proteins and plasmids encoding the capsid proteins are alsoprovided.

In one aspect, a capsid protein comprises an individual amino acid or anamino acid sequence from a first AAV serotype, and from at least asecond AAV serotype.

In one embodiment, the capsid protein additionally comprises a sequenceof amino acid residues from a contiguous sequence of amino acids from athird AAV serotype.

In another embodiment, the sequences of amino acids in the firstsequence, in the second sequence, and in the third or further sequence,are each a contiguous sequence of amino acids from the first AAVserotype, the second AAV serotype, the third and/or further AAVserotypes. In another embodiment, the contiguous sequence of amino acidsforms a conserved set of amino acid residues, the conserved set havingat least about 70% sequence identity, more preferably at least about80%, still more preferably at least about 85%, and still more preferablyat least about 90% or 95% sequence identity with the AAV serotype from acontiguous sequence in its respective AAV serotype.

In one embodiment, the first AAV serotype is AAV-2 and the second AAVserotype is AAV-8 or AAV-9.

In another aspect, a capsid protein comprises an amino acid sequencecomprising a first sequence of amino acid residues of a first AAVserotype, a second sequence of amino acid residues of a second AAVserotype, and a third sequence of amino acid residues of a third AAVserotype.

In one embodiment, the first AAV serotype is AAV-2, the second AAVserotype is AAV-8, and the third AAV serotype is AAV-9.

In a preferred embodiment, a capsid protein comprises an amino acidsequence having at least about 80% sequence identity to the amino acidsequence of SEQ ID NO: 1. In another embodiment, the capsid protein isencoded by a nucleotide sequence having at least about 80% sequenceidentity to the nucleotide sequence of SEQ ID NO: 2.

A viral particle comprising a capsid protein sequence as describedabove, is contemplated in another embodiment.

In another aspect, a plasmid comprising a sequence selected from thegroup consisting of (i) sequences having at least 80% sequence identityto SEQ ID NO:2 and (ii) SEQ ID NO: 2 is provided.

In yet another aspect, a recombinant AAV vector is provided, the vectorcomprising a capsid protein having an amino acid sequence selected fromthe group of sequences consisting of (i) sequences having at least 80%sequence identity to SEQ ID NO:1 and (ii) SEQ ID NO: 1.

In still another aspect, a method of expressing a gene of interest in amammal is provided. The method comprises introducing a recombinant AAVvector into a mammal, the recombinant AAV vector encoding for a gene ofinterest which is encapsidated into a capsid protein having an aminoacid sequence selected from the group of sequences consisting of (i)sequences having at least 80% sequence identity to SEQ ID NO:1 and (ii)SEQ ID NO:1.

In still another aspect, a method of generating a library of recombinantAAV plasmids is disclosed, the method comprising: isolating AAV capsidnucleotide sequences from two or more serotypes of AAV; digesting theAAV capsid nucleotide sequences into fragments; reassembling thefragments using PCR to form PCR products; and cloning the re-assembledPCR products into plasmids to generate a library of recombinant AAVplasmids.

In one embodiment, the method includes isolating AAV capsid nucleotidesequences from human AAV serotypes and non-human AAV serotypes.Exemplary serotypes include AAV-2, AAV-8, and AAV-9.

In another embodiment, the method comprises transfecting cells with theplasmids to produce a viral library, preferably an AAV viral library.

In one embodiment, the transfection includes transfecting into 293kidney cells with a helper Adenovirus.

In another embodiment, the method additionally includes, after thetransfecting, passaging the viral library in a selected cell type in thepresence of a stringent condition, and selecting AAV capsids thatsurvive the passaging. Passaging can be for several or multiplepassages, for example from between 2-5 or 2-10 passages.

In one embodiment, a stringent condition comprises the presence of humanimmune globulin.

In another aspect, a library prepared according to the methods describedabove is disclosed. In one embodiment the library is comprised ofplasmids of shuffled full-length capsid genes and in another embodimentthe library is comprised of viral particles obtained by transfecting allor a portion of the plasmid library into a selected cell, optionally incombination with an adenoviral helper plasmid.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B show an alignment of the amino and sequences ofAAV-DJ (SEQ ID NO: 1) and of the capsid proteins of AAV-2 (SEQ ID NO:3), AAV-8 (SEQ ID NO: 4), and AAV-9 (SEQ ID NO: 5);

FIGS. 2A-2C are graphs showing the infectious particles per mL of AAV-DJviral particles, AAV-2. AAV-8, and AAV-9 after neutralizing assays usinghuman immune globulin (IVIG) in 293 cells (FIGS. 2A, 2C), Huh-7 cells(FIG. 2B) at antiserum to virus dose ratios of 1:1 (FIGS. 2A-2B) or 1:2(high), 1:10 (med), and 1:25 (low) (FIG. 2C);

FIG. 3 is a bar graph showing green fluorescent protein (gfp)expression, in IU/mL, in human melamona cells in vitro followingtransduction with recombinant AAV-DJ particles or with wildtype AAV-1,AAV-2, AAV, 3, AAV-4, AAV-5, AAV-6, AAV-8, or AAV-9 particles thatexpress gfp;

FIGS. 4A-4C are graphs showing the amount of factor IX protein (ng/mL)in mice, as a function of days post-injection of AAV-DJ (circles), AAV-2(diamonds), AAV-8 (squares), or AAV-9 (triangles) expressing humanfactor IX (FIX) gene at doses of 5×10¹⁰ (FIG. 4A), 2×10¹¹ (FIG. 4B), and1×10¹² (FIG. 4C);

FIG. 5 is a bar graph showing the expression of humanalpha-1-antitrypsin (hAAT), in ng/mL, in mice injected with identicaldoses (2×10¹¹) of recombinant AAV-2, AAV-8, AAV-9, or AAV-DJ vectorsexpressing hAAT, the expression measured 3 (open), 7 (dotted) or 14(cross-hatched) days after injection;

FIGS. 6A-6B are graphs showing plasma hFIX levels in mice immunized with4 mg (FIG. 6A) or 20 mg (FIG. 6B) IVIG prior to injection ofhFIX-expressing AAV-DJ (open circles), AAV-2 (closed diamonds), AAV-8(closed squares), or AAV-9 (closed triangles) as a function of timepost-injection, the hFIX levels shown as a percent of the correspondinglevel in control mice treated with phosphate-buffered saline rather thanIVIG;

FIG. 6C is a bar graph showing the hFIX plasma concentration, in ng/mL,in mice injected with PBS or hAAT-expressing AAV-2, -8, -9 or -DJ (Xaxis), and three weeks later re-injected hFIX-expressing viruses, thehFIX plasma concentrations measured six weeks after the secondinjection;

FIG. 6D is a bar graph showing neutralizing antibody titers (NAb)against the wildtype AAVs or AAV-DJ in sera taken from the mice, treatedas described in FIG. 6C, at the time of re-injection (H), as well asfrom a parallel group injected with a lower dose (L) of 2×10¹⁰particles;

FIG. 7A shows amino acid residues at positions 585-588 in AAV-2 and themodifications at the two arginine (R) residues in AAV-2, AAV-8, AAV-9,or AAV-DJ mutagenized to eliminate or introduce a heparin bindingdomain;

FIG. 7B is a bar graphs showing the titration of infectious particles onkidney cells, in IU/mL for AAV-2, AAV-8, AAV-9, AAV-DJ, and for themutants (FIG. 7A) AAV-2/8, AAV-8/2, AAV-9/2, AAV-DJ/8, and AAV-DJ/9;

FIGS. 7C-7D are bar graphs of cells binding assays in HeLa (FIG. 7C) andHuh-7 (FIG. 7D) cells, showing the binding, expressed as a percentage ofAAV-2, of AAV-2, AAV-8, AAV-9, AAV-DJ, and for the mutants (FIG. 7A)AAV-2/8, AAV-8/2, AAV-9/2, AAV-DJ/8, and AAV-DJ/9;

FIG. 8 is a flow chart summarizing a method of generating a library ofAAV capsids;

FIG. 9 is a flow chart summarizing a method of isolating recombinantAAV.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is an amino acid sequence of a novel recombinant VP1 capsidprotein, referred to herein as AAV-DJ.

SEQ ID NO:2 is a nucleotide sequence encoding the protein AAV-DJ.

SEQ ID NO:3 is the amino acid sequence of the capsid protein of AAV-2.

SEQ ID NO:4 is the amino acid sequence of the capsid protein of AAV-8.

SEQ ID NO:5 is the amino acid sequence of the capsid protein of AAV-9.

SEQ ID NOS:6-15 are artificial primers.

DETAILED DESCRIPTION I. Definitions

The practice of the subject matter described herein will employ, unlessotherwise indicated, conventional techniques of molecular biology,microbiology, cell biology and recombinant DNA, which are within theskill of the art. See, e.g., Sambrook. Fritsch, and Maniatis, MOLECULARCLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS INMOLECULAR BIOLOGY, (F. M. Ausubel et al. eds., 1987); the series METHODSIN ENZYMOLOGY (Academic Press, Inc.); PCR 2: A PRACTICAL APPROACH (M. J.McPherson, B. D. Hames and G. R. Taylor eds., 1995) and ANIMAL CELLCULTURE (R. I. Freshney. Ed., 1987).

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus,the term polynucleotide sequence is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching.

An “isolated polynucleotide” molecule is a nucleic acid moleculeseparate and discrete from the whole organism with which the molecule isfound in nature; or a nucleic acid molecule devoid, in whole or part, ofsequences normally associated with it in nature; or a sequence, as itexists in nature, but having heterologous sequences in associationtherewith.

Techniques for determining nucleic acid and amino acid “sequenceidentity” also are known in the art. Typically, such techniques includedetermining the nucleotide sequence of the mRNA for a gene and/ordetermining the amino acid sequence encoded thereby, and comparing thesesequences to a second nucleotide or amino acid sequence. In general,“identity” refers to an exact nucleotide-to-nucleotide or aminoacid-to-amino acid correspondence of two polynucleotides or polypeptidesequences, respectively. Two or more sequences (polynucleotide or aminoacid) can be compared by determining their “percent identity.” Thepercent identity of two sequences, whether nucleic acid or amino acidsequences, is the number of exact matches between two aligned sequencesdivided by the length of the shorter sequences and multiplied by 100.Percent identity may also be determined, for example, by comparingsequence information using the advanced BLAST computer program,including version 2.2.9, available from the National Institutes ofHealth. The BLAST program is based on the alignment method of Karlin andAltschul. Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and asdiscussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); KarlinAnd Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); andAltschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, theBLAST program defines identity as the number of identical alignedsymbols (i.e., nucleotides or amino acids), divided by the total numberof symbols in the shorter of the two sequences. The program may be usedto determine percent identity over the entire length of the proteinsbeing compared. Default parameters are provided to optimize searcheswith short query sequences in, for example, blastp with the program. Theprogram also allows use of an SEG filter to mask-off segments of thequery sequences as determined by the SEG program of Wootton andFederhen. Computers and Chemistry 17:149-163 (1993). Ranges of desireddegrees of sequence identity are approximately 80% to 100% and integervalues therebetween. Typically, the percent identities between adisclosed sequence and a claimed sequence are at least 80%, at least85%, at least 90%, at least 95%, or at least 98%.

Alternatively, the degree of sequence similarity between polynucleotidescan be determined by hybridization of polynucleotides under conditionsthat form stable duplexes between homologous regions, followed bydigestion with single-stranded-specific nuclease(s), and sizedetermination of the digested fragments. Two DNA, or two polypeptidesequences are “substantially homologous” to each other when thesequences exhibit at least about 80-85%, preferably 85-90%, morepreferably 90-95%, and most preferably 98-100% sequence identity to thereference sequence over a defined length of the molecules, as determinedusing the methods above. As used herein, substantially homologous alsorefers to sequences showing complete identity to the specified DNA orpolypeptide sequence. DNA sequences that are substantially homologouscan be identified in a Southern hybridization experiment under, forexample, stringent conditions, as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic AcidHybridization, supra.

II. Chimeric AAV Capsid

In one aspect, capsid proteins with regions or domains or individualamino acids that are derived from two or more different serotypes of AAVare provided. In one embodiment, described below, a capsid proteincomprised of a first region that is derived from a first AAV serotype, asecond region that is derived from a second AAV serotype, and a thirdregion that is derived from a third AAV serotype is provided. The AAVserotypes may be human AAV serotypes or non-human AAV serotypes, such asbovine, avian, and caprine AAV serotypes. In particular, non-primatemammalian AAV serotypes, such as AAV sequences from rodents (e.g., mice,rats, rabbits, and hamsters) and carnivores (e.g., dogs, cats, andraccoons), may be used. By including individual amino acids or regionsfrom multiple AAV serotypes in one capsid protein, capsid proteins thathave multiple desired properties that are separately derived from themultiple AAV serotypes may be obtained.

In one embodiment, a capsid protein, referred to herein as “AAV-DJ”,that has an amino acid sequence comprising a first region that isderived from a first AAV serotype (AAV-2), a second region that isderived from a second AAV serotype (AAV-8), and a third region that isderived from a third AAV serotype (AAV-9), is provided. The AAV-DJcapsid protein was identified from a library of capsid proteins, thelibrary generated using a method described below (Example 1). It will beappreciated that the AAV-DJ protein is merely exemplary of thebeneficial capsid proteins that can be obtained from a library generatedaccording to the teachings herein, where the beneficial capsid proteinspreferably have multiple desired properties that are derived frommultiple AAV serotypes.

The amino acid sequence of AAV-DJ is shown in SEQ ID NO: 1, and thenucleotide sequence encoding AAV-DJ is shown in SEQ ID NO: 2. FIGS. 1Aand 1B show an alignment of the amino acid sequences of AAV-DJ and ofthe capsid proteins of AAV-2 (SEQ ID NO:3), AAV-8 (SEQ ID NO:4), andAAV-9 (SEQ ID NO:5). The five boxes numbered 1-5 in FIGS. 1A and 1Brepresent the five known loops on the exterior of the AAV-2 capsid whichare likely to be involved in capsid binding to cellular receptors andrecognized by neutralizing antibodies. The alignment in FIGS. 1A and 1Bshow that the N-terminus of AAV-DJ is identical to the N-terminus of theAAV-2 capsid protein and that the C-terminus of AAV-DJ is identical tothe C-terminus of the AAV-8 capsid protein. The loop 1 region of AAV-DJis identical to the loop 1 region of AAV-9. The loop 2, 3, and 5 regionsof AAV-DJ are identical to the corresponding regions of AAV-8. The loop4 region of AAV-DJ is a hybrid of the loop 4 regions of AAV-2 and AAV-8,with parts of the AAV-DJ loop 4 region being identical to parts of theloop 4 region of AAV-2, parts of the AAV-DJ loop 4 region beingidentical to parts of the loop 4 region of AAV-8, and parts of the loop4 region of AAV-DJ being identical to both parts of the loop 4 region ofAAV-2 and of AAV-8.

AAV-DJ has four mismatches to the two T cell epitopes in AAV-2 whichhave recently been identified as being involved in an anti-AAV cytotoxicT lymphocyte (CTL) response in humans. Thus, recombinant AAV vectorsthat include the AAV-DJ capsid protein or a derivative thereof arelikely less immunogenic in humans than AAV-2 vectors that include theAAV-2 capsid.

Studies were conducted to confirm that infectious viral particles can beformed with AAV-DJ as the capsid. In a first study, the AAV-DJnucleotide sequence was inserted into an AAV helper plasmid that alsoexpresses the AAV-2 rep gene (Example 2). 293 kidney cells were thenco-transfected with the AAV helper plasmid and an adenoviral helperplasmid, as well as a gfp-expressing vector plasmid. For comparison, twodifferent versions of an AAV-2 helper were used (designated AAV-2 “old”and AAV-2 “new”) which differ in the expression levels of viralproteins. Three days after the co-transfection, Western blotting (with303.9 (Rep) and B1 (capsid protein)) of the 293 cell extracts revealedthe presence of presence of Rep and capsid proteins at levels comparableto those found in cells co-transfected with plasmids expressing theAAV-2, AAV-8, or AAV-9 capsid proteins (blot not shown).

In another study, particle infectivity and ability to avoidneutralization by human immune globulin (IVIG) of AAV-DJ clone wascompared to wildtypes AAV-2, AAV-8, and AAV-9. Two different versions ofan AAV-2 helper were used (designated AAV-2 old and AAV-2 new) whichdiffer in the expression levels of viral proteins. Recombinant AAVs witheither the AAV-DJ, AAV-2, AAV-8, or AAV-9 capsids were produced bytriple transfecting cells with a plasmid encoding gfp flanked by AAVinverted terminal repeats (ITRs), a plasmid encoding adenoviral helpergenes, and a plasmid encoding the AAV-2 Rep gene and either the AAV-DJ,AAV-2, AAV-8, or AAV-9 capsid protein, and then freeze-thaw lysing thecells. Each virus-containing lysate was then neutralized using a highdose (1:1 volume) of two different batches of human immune globulin(IVIG1 and IVIG2) (FIG. 2A (293 cells); FIG. 2B (Huh-7 cells)), or threedecreasingly lower doses (1:2 (high), 1:10 (med), and 1:25 (low)antiserum/virus) of the two different batches of human immune globulin(IVIG1 and IVIG2), or a monoclonal A20 antibody (FIG. 2C, 293 cells), ora polyclonal anti-AAV-8 serum (“A8”). A20 is a monoclonal antibody thatwas raised against assembled AAV-2 capsids and anti-AAV-8 is apolyclonal rabbit serum raised against assembled AAV-8 capsids. Lysatestreated with PBS were used as a control. The virus-containing lysateswere neutralized by incubating the lysates with the human immuneglobulin or antibody for a period of time (one hour at room temperature(20-25° C.)) and then infecting cells in the presence of helperadenovirus. The remaining activity of the viruses after theneutralization period was determined by titrating gfp expression unitson the cells.

The results for the 293 cells are shown in FIG. 2A and for the Huh-7cells in FIG. 2B. In the absence of IVIG1, IVIG2, and A20, the AAV-DJvirus was at least as infectious on 293 cells as AAV-2 and several foldmore infectious than AAV-2 on Huh-7 cells. The data also shows that theAAV-DJ virus and AAV-8 were able to partially escape neutralization byIVIG, while AAV-2 was not. AAV-9 had intermediate IVIG results relativeto AAV-DJ/AAV-8 and AAV-2, and was neutralized at high IVIG doses. AAV-2was neutralized by the A20 antibody, but the A20 antibody did notsignificantly affect AAV-DJ, AAV-8, or AAV-9. The polyclonal anti-AAV-8antiserum neutralized all four capsids at high or medium doses, whereasAAV-2 and AAV-DJ partially escaped neutralization at the low dose.

In summary, it was found that the AAV-DJ virus was more infectious toHuh-7 cells than the previously known most efficient AAV on Huh-7 cells(AAV-2) even in the presence of high concentrations of human immuneglobulin. Also, the AAV-DJ virus was found to have improved resistanceto neutralization by human immune globulin relative to AAV-2. Suchresistance is reasonable, given that the AAV-DJ capsid was selected froma library partially based on its ability to produce virus that resistneutralization by human immune globulin. However, the improvedresistance of the AAV-DJ virus to the A20 antibody was surprising andunexpected, because (i) it was not part of the selection schemedescribed below that was used to isolate AAV-DJ; and (ii) AAV-DJ sharessubstantial identity to AAV-2, which is neutralized by the A20 antibody.

In yet another study using human melanoma cell, in vitro infectivity ofgfp-expressing vectors from the AAV-DJ capsid gene was compared to thein vitro infectivity of eight commonly used wildtype AAVs, AAV-1, AAV-2,AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, or AAV-9. The melanoma cells wereinfected with 2×10⁹ recombinant AAV particles of each serotype and gfpexpression was visualized three days later. The quantative results,expressed as gfp expression in IU/mL, from virus titration on themelanoma cells (in 96-well plates) are shown in FIG. 3. The AAV-DJvector was superior to the wildtype vectors, and, notably, substantiallybetter than AAV-2.

A number of cell lines were infected with ten-fold serial dilutions ofeach serotype, or AAV-DJ or the DJ heparin mutant DJ/8, discussed below,expressing a gfp reporter gene. Vector preparations were normalized tocontain 2×10⁹ total (vector DNA-containing) particles per mL prior toinfection. Three days later, gfp-expressing cells were counted andinfectious titers determined, taking into account the dilution factor.As seen in Table 1, AAV-DJ vectors showed the highest infectivity on alltested cell lines, and ratios of total to infectious particles werefrequently far below 500, highlighting the extreme efficiency of AAV-DJin vitro, and suggesting its particular usefulness for ex vivo genetransfer applications.

TABLE 1 In vitro infectivity of AAV-DJ and wildtype vectors Ratio ofTotal to Infectious AAV particles¹ (×10³) Cell AAV AAV AAV AAV AAV AAVAAV AAV AAV AAV line Tissue² 1 2 3 4 5 6 8 9 DJ DJ/8 Huh-7 hu liver 40.5 20 2000 400 5 70 7000 0.1 300 293 hu kidney 2 0.5 20 700 400 10 70700 0.1 200 HeLa hu cervix 70 2 100 2000 30 200 1000 2000 0.3 1000 HepG2hu liver 2000 50 300 20000 3000 1000 20000 nd 4 10000 Hep1A mu liver 102 1000 200 2000 200 1000 20000 0.5 2000 911 hu retina 6 1 9 500 700 60001000 nd 0.2 400 CHO ha ovary 10 10 70 700 3 20 100 1000 0.04 200 COS sikidney 3 1 3 30 20 7 50 200 0.2 300 MeWo hu skin 2 0.2 1 70 3 2 20 1000.007 20 NIH3T3 mu fibrobl. 200 20 700 700 7000 200 7000 nd 4 20000 A549hu lung 70 10 50 nd 2000 100 2000 7000 1 20000 HT1180 hu fibrobl. 50 10100 7000 3000 30 2000 10000 3 5000 ¹Numbers shown are average ratios(rounded) of total to infectious AAV particles from at least threeindependent titrations. Lower numbers indicate higher infectivity. ²hu,human; mu, murine; ha, hamster; si, simian; fibrobl., fibroblasts; nd,not detectable (>2 × 10⁷).

Vectors prepared with the AAV-DJ capsid were also tested in vivo forexpression of a gene of interest. In a first study, recombinant humanfactor IX (FIX)-expressing AAVs with either the AAV-DJ, AAV-2, AAV-8, orAAV-9 capsids were produced by a triple transfection technique describedin Example 3. Doses of 5×10¹⁰, 2×10¹¹, and 1×10¹² (low, medium, andhigh, respectively) recombinant viral particles were injectedperipherally into immunocompetent mice (C57/BL6) and plasma hFIX wasmonitored for up to four months after injection. The FIX protein plasmalevels were quantified by ELISA, and the results are shown in FIGS.4A-4C.

In FIGS. 4A-4C, the shading represents 1-100% normal hFIX levels inhumans (0.05 to 5 μg/mL). FIX levels over 1% are considered therapeuticin hemophilics. As seen, the AAV-8, -9 or -DJ vectors exceeded the 100%level already at the lowest dose. A dose-dependent expression from theAAV-DJ capsid at levels equivalent to AAV-8 and -9, the best twonaturally identified AAVs reported in liver thus far, was observed. Allthree viruses readily outperformed the AAV-2 prototype at any dose andexpressed over 100% of normal hFIX levels from intravenous injection of5×10¹⁰ particles, whereas AAV-2 expression was over 100% of normal hFIXlevels only at a dose of 1×10¹².

In another study, recombinant human alpha-1-antitrypsin(hAAT)-expressing AAVs were prepared, from the AAV-DJ, AAV-2, AAV-8, orAAV-9 capsids. The hAAT gene was under an RSV promoter. Mice (C57/BL6)were injected via tail vein infusions of 2×10¹¹ particles and plasmalevels of hAAT were determined via specific ELISA 3, 7, and 14 daysafter injection. Results are shown in FIG. 5. AAV-8, AAV-9, and AAV-DJexpressed efficiently and equally outperformed the vector with an AAV-2capsid.

In another in vivo study, liver transduction in the presence of humanserum was quantified, to assess the ability of AAV-DJ to evadeneutralization in vivo. As described in Example 4, mice were passivelyimmunized with 4 or 20 mg IVIG prior to infusion of hFIX-expressingAAV-2, -8, -9, or -DJ. Plasma hFIX levels for each AAV serotype areshown in FIGS. 6A-6B as percent corresponding virus level in controlmice treated with phosphate-buffered saline rather than IVIG as afunction of time post infusion. FIG. 6A shows the results for miceimmunized with 4 mg IVIG and FIG. 6B shows the results for miceimmunized with 20 mg IVIG. AAV-2 expression was completely abolished,however transduction with AAV-DJ, -8 or -9 was inhibited in adose-dependent manner, with AAV-DJ showing intermediate resistance atthe high, and efficient evasion (similar to AAV-8 and AAV-9) at the lowIVIG dose (FIG. 6A). These results were confirmed with a secondindependent IVIG batch from another vendor (Carimune 12%, Behring AG,data not shown).

In another study, also described in Example 4, the feasibility torepeatedly administer the different viruses to mice was assessed, toevaluate capsid cross-neutralization. Results are shown in FIG. 6C. Nogene expression upon re-infusion of any of the capsids into animalsalready treated with the same serotype was observed. However, AAV-8 and-9 also efficiently blocked each other, substantiating previous data(Gao. G. et al., J. Virol., 78:6381-6388 (2004)). This result mightargue against the use of vectors based on these wildtypes inre-administration protocols, albeit they could be combined with AAV-2.In contrast, primary infusion of AAV-DJ allowed subsequent expression(up to 18%) from AAV-2, -8 or -9, likely due to the fact that AAV-DJonly shares a limited number of epitopes with each wildtype virus. Inthe reverse experiment. AAV-DJ vectors were inhibited in animalsimmunized with AAV-8 or -9, while giving detectable expression inAAV-2-treated mice. This implied a stronger or broader immune responsefrom primary infusion of serotypes 8 or 9. AAV-DJ was more resistant tothe corresponding mouse sera in culture, as seen in FIG. 6D. Lesscross-reactivity between AAV-8 and -9 was noted.

AAV-DJ, as well as other recombinant protein capsids identified in thelibrary discussed below, retained a heparin binding domain (HBD) fromthe AAV-2 parent. This domain functions in binding to the primary AAV-2receptor heparin sulfate proteoglycan (Summerford. C. et al., J. Virol.,72:1438-1445 (1998)). To investigate the role of the AAV-DJ HBD, twocrucial arginine residues (Kern, A. et al., J. Virol., 77:11072-11081(2003)) were mutated to the respective residues in AAV-8 or -9, as shownin FIG. 7A, and are referred to herein as AAV-DJ/8 and AAV-DJ/9. Table 1above includes data on the mutant AAV-DJ/8, and shows that gfpexpression was reduced by several orders of magnitude, and was as low asthat observed with serotypes AAV-8 or AAV-9.

The infectivity drop (Table 1) was shown to correlate with a reducedbinding to cells. As seen in FIG. 7B, a titration of infectiousparticles on 293 kidney cells illustrated the role of the HBD forinfection in culture, as seen by the reduction in infectivity in the HBDmutants AAV-DJ/8 and AAV-DJ/9. Additional mutants were prepared andtested, and are identified herein as AAV-2/8 (HBD negative), AAV-8/2(HBD positive), and AAV-9/2 (HBD positive). Cell binding assays, shownin FIGS. 7C-7D, confirmed the role of the HBD for attachment to culturedcells, where the drop in binding with the mutants correlated with thetransduction data in FIG. 7B. The HBD-positive AAV-8 and AAV-9 mutantsbound several fold more efficiently than AAV-2 on HeLa cells (FIG. 7C),but transduced less efficiently. Thus, cell attachment alone cannotexplain the unusual infectivity of AAV-DJ. Instead, a synergistic effectfrom sharing beneficial properties from all AAV-DJ parents iscontemplated, resulting in enhancement of multiple steps in AAV-DJtransduction.

The HBD also was shown to influence biodistribution, as shown in Table2. AAV-8 and -9 (HBD-negative) demonstrated an unrestricted tropism,readily transducing all tested tissues at 1×10¹² particles per mouse. Instriking contrast, AAV-2 and likewise AAV-DJ (both HBD-positive) wererestricted to liver and, to a lesser extent, heart, kidney and spleen,and near or below detection limit in all other tissues. Quantificationof double-stranded vector DNA (using liver as an internal standard ineach group) showed that AAV-DJ transduced lung, brain, pancreas and gutabout 2- to 4-fold less efficiently than wildtypes 8 or 9. The effect ofthe HBD on viral tropism was best exemplified by comparing AAV-DJ to theDJ/8 mutant: HBD deletion alleviated the liver restriction and expandedtransduction to all nonhepatic tissues, identical to AAV-8 and -9, andincluding the brain. These findings corroborate and explain a series ofreports on wide tissue dissemination of vectors based on HBD-negativenatural serotypes (AAV-1 and -4 to -9) in mice, dogs and monkeys, incontrast to the HBD-positive AAV-2. Notably, AAV-DJ also transducednonhepatic tissues at the maximum dose of 7×10¹² particles, but still toa lesser extent than the HBD-negative viruses, in particular AAV-9. Evenat this dose, brain and also lung transduction remained marginal.

TABLE 2 Relative transduction of non-hepatic tissues with AAV vectors

Vector copy numbers (per diploid genomic equivalent) were determined viaPhosphoimager scan analyses of Southern Blots. At least threeindependent mice were anlaysed per dose. Copy numbers are shown inpercent (rounded to one decimal, plus standard deviations) relative tothose in liver within each group, allowing comparison between vectorsand doses. For AAV-2, most signals were below the detection limit of theSouthern Blot analyses (~0.03 copies of double-stranded AAV DNA percell), preventing calculation of relative transduction in these cases(nd, not determined). Gray shadows highlight doses/tissues whererelative AAV-DJ transduction differed by at least 2-fold from serotypes8 and 9, as well as the AAV-DJ HBD mutant.

While the embodiments described above are primarily with respect to anAAV-DJ capsid having the amino acid sequence of SEQ ID NO: 1 and thenucleotide sequence of SEQ ID NO: 2, it is recognized that capsidshaving amino acid and/or nucleotide sequences that are similar insequence to SEQ ID NO: 1 and SEQ ID NO: 2 and having the same functionmay be used and are contemplated. In one embodiment, a recombinantcapsid protein having at least about 60% identity, further at leastabout 70% identity, preferably at least about 80% identity, morepreferably at least about 90% identity, and further preferably at leastabout 95% identity, to the amino acid sequences identified as SEQ IDNO:1 is contemplated.

It will be appreciated that conservative amino acid substitutions may beto the protein of SEQ ID NO:1, to achieve proteins having, for example,60%, 70%, 80%, 90%, or 95% sequence identity to SEQ ID NO:1, andpreferably with retention of activity of the native sequence.Conservative amino acid substitutions, as known in the art and asreferred to herein, involve substituting amino acids in a protein withamino acids having similar side chains in terms of, for example,structure, size and/or chemical properties. For example, the amino acidswithin each of the following groups may be interchanged with other aminoacids in the same group: amino acids having aliphatic side chains,including glycine, alanine, valine, leucine and isoleucine; amino acidshaving non-aromatic, hydroxyl-containing side chains, such as serine andthreonine; amino acids having acidic side chains, such as aspartic acidand glutamic acid; amino acids having amide side chains, includingglutamine and asparagine; basic amino acids, including lysine, arginineand histidine; amino acids having aromatic ring side chains, includingphenylalanine, tyrosine and tryptophan; and amino acids havingsulfur-containing side chains, including cysteine and methionine.Additionally, amino acids having acidic side chains, such as asparticacid and glutamic acid, are considered interchangeable herein with aminoacids having amide side chains, such as asparagine and glutamine.

In one embodiment, the recombinant AAV capsid protein is comprised of afirst sequence of amino acid residues from a first AAV serotype, and atleast a second sequence of amino acid residues from a second AAVserotype. The first sequence is, in the embodiment, a conserved set ofamino acids from a contiguous sequence of amino acids from the first AAVserotype. The second sequence is a conserved set of amino acids from acontiguous sequence of amino acids from the second AAV serotype. A“conserved set” of amino acids refers to a contiguous sequence of aminoacids that is identical or closely homologous to a sequence of aminoacids in the AAV serotype. In one embodiment, close homology intends atleast about 80% sequence identity. A contiguous sequence of amino acidsin such a conserved set may be anywhere from 2 to 500, 2 to 400, 2 to300, 2 to 200, 2 to 100, or 2 to 50 amino acid residues in length.

It will also be appreciated that the recombinant vectors describedherein are contemplated for use in methods of expressing a gene ofinterest in a variety of cells and in a mammal. Transduction into cellslines in addition to the cell lines described herein, for example inTable 1, are exemplary, and other cells lines, particularly stem cells,are contemplated. In terms of in vivo use, the method preferablycomprises introducing a recombinant AAV into the mammal, the recombinantAAV encoding the gene of interest and comprising a capsid protein havingan amino acid sequence selected from the group of sequences consistingof (i) sequences having 80% sequence identity to SEQ ID NO:1 and (ii)SEQ ID NO: 1. The vector expressing a gene of interest is introduced tothe mammal, typically by injection, intravenously, subcutaneously,parenterally, or the like. The gene of interest can be any gene, andmany suitable genes for expression for therapeutic or non-therapeuticpurposes are readily identified by a skilled artisan. The nucleotidesequence of the gene of interest is typically “operably linked” to oneor more other nucleotide sequences, including but not limited to thegene for a selected capsid protein, a promoter, and enhancer, and thelike.

A gene is “operably linked” to another nucleotide sequence when it isplaced in a functional relationship with another nucleotide sequence.For example, if a coding sequence is operably linked to a promotersequence, this generally means that the promoter may promotetranscription of the coding sequence. Operably linked means that the DNAsequences being linked are typically contiguous and, where necessary tojoin two protein coding regions, contiguous and in reading frame.However, since enhancers may function when separated from the promoterby several kilobases and intronic sequences may be of variable length,some nucleotide sequences may be operably linked but not contiguous.Additionally, as defined herein, a nucleotide sequence is intended torefer to a natural or synthetic linear and sequential array ofnucleotides and/or nucleosides, and derivatives thereof. The terms“encoding” and “coding” refer to the process by which a nucleotidesequence, through the mechanisms of transcription and translation,provides the information to a cell from which a series of amino acidscan be assembled into a specific amino acid sequence to produce apolypeptide.

III. Generation of a Library of Novel AAV Capsids

In another aspect, a method of generating a library of novel AAV capsidsis provided. Embodiments of this aspect include a method of isolating arecombinant AAV plasmid that includes a novel AAV capsid. Theseembodiments will now be discussed with reference to FIGS. 8-9.

FIG. 8 summarizes a method of generating a library of novel AAV capsids.As shown in step 402 of FIG. 8, isolated nucleic acids encoding capsidgenes are obtained from multiple AAV serotypes (two or more) usingprimers designed to include a serotype-specific part fused with commonsignature regions that flank the capsid nucleic acid sequence. Then, asshown in step 404, the isolated nucleic acids are digested orfragmented, such as with DNAsel, into fragments of, for example, betweenabout 0.2 and about 1.0 kb. The fragments are then re-assembled in step406 into larger pieces by performing PCR, such as with Taq polymerase,in the absence of additional primers. Because of the related nature ofthe fragmented genes, the gene fragments have overlapping regions ofhomology that allow the fragments to self prime in the absence ofadditional primer. After multiple rounds of PCR, products having alength approximately equal to that of the originally capsid genes areobtained. The PCR products include hybrid products that contain capsidregions from multiple AAV serotypes.

As shown in step 408, the full length PCR products are then PCRamplified, preferably with Platinum Pfx polymerase, using primers thatbind to the signature regions that are contained in the full length PCRproducts because they were present in the original primers used toisolate the capsid nucleic acid sequences. The PCR products from step408 are then cloned into a conventional plasmid, as shown in step 410 toprovide a library of novel AAV capsid genes. In one embodiment, thecapsid genes are cloned into an ITR-rep-containing AAV plasmid, tosubsequently create the actual viral library.

FIG. 9 summarizes a method of isolating a recombinant AAV that includesa novel recombinant AAV capsid, i.e., a “hybrid capsid” is isolated asdescribed above with respect to FIG. 8. In step 502, hybrid capsidsequences are cloned into a plasmid that is capable of producing aninfectious AAV genome, such as a plasmid comprising the AAV-2 rep gene,as well as the two AAV-2 ITRs. In step 504, the plasmid library istransfected into cells together with an adenoviral helper plasmid toproduce virus. In step 506, the virus is then amplified in cells in thepresence of a helpervirus, such as wildtype Adenovirus-5 helpervirus.The virus may be amplified in the presence of one or more forms ofselective pressure, such as in the presence of human immune globulin.The viruses that survive multiple passages under the selective pressureare chosen for further study or use, as shown in step 508.

In a supporting study (Example 1), the approach outline in FIGS. 8-9 wasused to generate a library. In brief, the capsid gene from eightdifferent AAV serotypes (AAV-2, AAV-4, AAV-5, AAV-8, AAV-9, avian AAV,bovine AAV, and caprine AAV) was fragmented, and the PCR products fromstep 406 were blunt cloned into the pCR4-TOPO plasmid, available fromInvitrogen. Twenty-four (24) subclones were sequenced to confirm thatcapsid sequences that are a hybrid of different serotypes were created.Sequences from all eight of the serotypes were represented in thesubclones. Typically, the hybrid capsid sequences included sequencesfrom at least two, and often, more than six, of the serotypes. Thecapsid sequences in the pCR4-TOPO plasmid were then subcloned into aplasmid comprising the AAV-2 rep gene, as well as the two AAV-2 ITRs,that was then used to transform bacteria. It is estimated thatapproximately a library of 3×10⁴ hybrid AAV capsid gene variants wereobtained from a single reaction and from 10 plates of bacteria.Up-scaling (including plating on 100 plates of bacteria) resulted in aplasmid library of approximately 6.9×10⁵ clones. This plasmid librarywas then co-transfected into 293 human embryonic kidney cells togetherwith an adenoviral helper plasmid, to produce a viral library of hybridAAV particles.

This library of AAV capsid variants was then co-infected with wildtypeAdenovirus-5 helpervirus and successfully amplified in several celllines, including human kidney 293 cells, human hepatoma Huh-7 cells, andmouse fibroblast NIH3T3 cells. Successful amplification of the virallibrary was confirmed by Western blots of whole cell extracts using theB1 antibody which recognizes an eight amino acid epitope that is largelyconserved over most known AAV serotypes, and thus should be present inthe majority of the hybrid AAVs described herein. Replicating AAVparticles were detected in all of the tested cell lines for up to fiveconsecutive passages. Whole freeze-thaw cell extracts were used forinfecting fresh cells each time. To date, the viral library has alsobeen successfully passaged six times in primary human hepatocytes, whichare notoriously difficult to infect with vectors based on wildtype AAVs.

The viral library was also amplified in human Huh-7 cells in thepresence of human immune globulin (IVIG). It was found that the specificIVIG used (IVIG Gamimune®N 10% from Bayer) contained abundantneutralizing antibodies against AAV-2 and AAV-3, as well as someantibodies against AAV-1, AAV-4, AAV-5, and AAV-6. Thus, amplificationin human Huh-7 cells in the presence of IVIG provided a selectivepressure for AAV hybrids comprising domains from different serotypessince selecting for a high efficiency infection of Huh-7 cells favorsAAV-2 domains, while selecting for escape from IVIG neutralizationfavors AAV-8 and AAV-9 domains. The selection was successful, as it wasfound that with increasing passages of the library, an increasingtolerance to IVIG was achieved. After the fourth passage, survivingvirus could be amplified in the presence of 500 μL IVIG, while after thefirst passage, surviving virus could only be amplified in the presenceof approximately 10 μL IVIG.

After the 5^(th) passage, the hybrid capsid sequences were PCR amplifiedand blunt cloned in pCR4-TOPO. The capsid sequences from 96 colonieswere sequenced and found to be identical. The hybrid capsid sequence isthe AAV-DJ sequence described above.

In summary, a plasmid library was created using DNA Family Shuffling(Crameri, et al., Nature, 391: 288-291 (1998)) of parental AAV capsidgenes. Subsequently, a viral library was generated, by transfecting theplasmid library into cells together with an adenoviral helper plasmid.This second viral library was then subjected to selection pressure, toisolate specific candidates. From those, selected shuffled capsid geneswere isolated and subcloned into an AAV helper plasmid, to makerecombinant AAV vectors comprising the hybrid capsid. More particularly,DNA Family shuffling was used to create a complex library of hybridparticles from eight different wildtypes. Serial amplification on humancells enriched hybrids from a multitude of AAV serotypes, typicallycontaining an AAV-2 heparin binding domain (HBD). More stringentselection with pooled human antisera yielded a single AAV-2-8-9 chimera,referred to herein as AAV-DJ. Recombinant AAV-DJ vectors were superiorto natural AAVs in cultured cells and outperformed the AAV-2 prototypein tissue in vivo. Vectors with an AAV-DJ capsid were superior in vitroand gave a robust and specific in vivo performance, and provided anability to evade humoral neutralization by human serum.

IV. Examples

The following examples are illustrative in nature and are in no wayintended to be limiting.

Example 1 AAV Capsid Library Generation

A. Plasmids for AAV Capsid Library Generation

Plasmids containing full-length capsid (cap) genes of seven differentAAV serotypes were obtained (AAV-2, -4, -5, -8, -9, avian and bovineAAV). Goat AAV was partly synthesized (GeneArt, Regensburg, Germany) asa 888 nt fragment (nt 1023 to 1910). This subclone spans the entireright half of the goat AAV capsid protein, which comprises all 42reported differences between goat AAV and AAV-5. The other seven capgenes were initially amplified via PCR and subcloned into pBlueScript IISK (Stratagene). The purpose was to flank all cap genes with sites forthe unique restriction enzymes Pac I (5′) or Asc I (3′), to facilitatelater cloning of “shuffled” cap genes into a wildtype AAV plasmid (seebelow). All primers also contained either a Hind III (5′) or a Spe I(3′) site, to allow directed cloning into pBlueScript (none of the fourrestriction enzymes cuts in any parental cap gene). A 20 nt signatureregion was inserted between the two restriction sites in each primer, toprovide conserved primer binding sites for later PCR amplification ofshuffled genes. Together, the sequence of the forward primers was

(SEQ ID NO: 6 5′ GGACTC AAGCTT GTCTGAGTGACTAGCATTCG

 CAGGT ATG 3′;Hind III site in bold, Pac I site in italics/bold, signature regionunderlined) directly attached at the 3′ end to the first 22 nt of eachcap gene following its ATG start codon. Likewise, the reverse primer was5′ CGTGAG ACTAGT GCTTACTGAAGCTCACTGAG GGCGCGCC TTA 3′ (SEQ ID NO:7; SpeI site in bold, Acs I site in italics/bold, signature region underlined)directly attached at the 3′ end to the last 22 nt of each cap gene up tothe TAA stop codon.

In parallel, a wildtype cap recipient plasmid was engineered to containthe AAV-2 packaging elements (ITRs) flanking the AAV-2 rep gene(encoding AAV replication proteins), together with Pac I and Asc I sitesfor cap cloning, and the AAV-2 polyadenylation site. Therefore. AAV-2rep (nt 191 to 2189) was PCR amplified using primers containing Bgl IIsites and then subcloned into pTRUF3 (carrying AAV-2 ITRs with adjacentBgl II sites). The forward primer used was 5′ CGAACC AGATCTGTCCTGTATTAGAGGTCACGTGAG 3′ (SEQ ID NO:8; Bgl II site in bold. AAV-2 nt191 underlined), and the reverse primer was

(SEQ ID NO: 9 5′ GGTAGC AGATCT GTTCGACCGCAGCCTTTCGAATGTCCGG TTTATTGATTA 

 CTGGACTC  TTAATTAA CATTTATTGTTCAAAGATGC 3′;Bgl II site in bold, polyadenylation signal underlined, Asc I site initalics/bold, Pac I site in italics/bold/underlined. AAV-2 rep stopcodon in italics/underlined). Note that this changed the AAV-2 Swa Isite (downstream of rep stop codon) into a Pac I site.

B. DNA Family Shuffling of AAV Capsid Genes

For DNA shuffling of AAV capsid genes, a 2-step protocol was used wherethe parental genes were first fragmented using DNase I enzyme and thenreassembled into a full-length gene via primer-less PCR. This wasfollowed by a second PCR including primers binding outside of the capgenes, allowing their subcloning into the wildtype recipient ITR/repplasmid. Initially, all cap genes were isolated from the subclones viaHind III/Spe I digestion (Eco RI for goat AAV) and then reactionconditions were optimized as follows. Various DNAse I concentrations andincubation times were tested, aiming to obtain a pool of fragmentsbetween 0.2 and 1.0 kb in size. Optimal conditions found were: 1 μg percap gene, 1 μL 1:200 pre-diluted DNase I (10 U/μL, Roche), 50 mM Tris ClpH 7.4, 1 mM MgCl₂, total volume of 50 μL. The reaction was incubatedfor 2 min at room temperature and then stopped by heat inactivating at80° C. for 10 min. Fragments of the desired sizes were isolated byrunning the entire reaction on a 1% agarose gel (total final volume ˜60μl). The re-assembly PCR reaction was then optimized by testing variousDNA polymerases (Pfx Platinum, Stratagene; DeepVent, NEB; Taq, Amersham)and respective conditions. Best results were obtained using PuReTaqReady-To-Go PCR Beads (Amersham) and the following conditions: 25 μLpurified cap fragments, program: 4 min 95° C., 40 cycles (1 min 95° C.,1 min 50° C., 3 min 72° C.), 10 min 72° C., 10 min 4° C. Agarose gel(1%) analysis of 1 μL from this reaction typically showed a smear up to5 kb and no distinct bands. The same three polymerases as above werethen evaluated for the primer-containing second PCR, and the followingconditions were found optimal: 1 μL Pfx Platinum, 2 μL product fromfirst PCR, 1 mM MgSO4, 1 μg of each primer (see below), 0.3 mM eachdNTP, total volume 50 μL, program: 5 min 94° C., 40 cycles (30 sec 94°C., 1 min 55° C., 3 minutes 68° C.), 10 min 68° C., 10 min 4° C. Theprimers used bound to the 20 nt signature regions described in theprevious chapter. This reaction gave a distinct ˜2.2 kb full-length capband (1% agarose gel), which was purified (60 μL total) and cloned (4μL) using the Zero Blunt TOPO PCR cloning kit (with electro-competentTOP10 cells) (Invitrogen, Carlsbad, Calif., USA). This intermediatecloning step significantly enhanced the yield of shuffled cap genes, ascompared to efforts to directly clone the PCR product via conventionalmeans (data not shown). The shuffled cap genes were then released fromthe TOPO plasmid via Pac I and Asc I double digestion and cloned intothe appropriately digested ITR/rep recipient plasmid. Performing allthese reactions under minimal conditions (volumes and amounts), alibrary of approximately 3×10⁴ bacterial colonies was obtained.Up-scaling of each step (including final plating on 100×15 cm plates)resulted in a final library of ˜6.9×10⁵ plasmid clones. Its integrity,genetic diversity and functionality was confirmed by DNA sequencing andsmall scale expression studies. From the latter, it was determined byextrapolation that the viral library (below) retained >90% viability.

C. Selective In Vitro Amplification of the Capsid Library

A viral library was prepared by transfecting 50×T225 flasks of 293 cellswith 50 μg plasmid per flask from the bacterial library, together with25 μg of an adenoviral helper plasmid. The resulting hybrid viruses wereconcentrated, purified and titrated as described for recombinant AAV.The final library had a particle titer (viral genomes) of 8.2×10¹¹/mL.Various amounts of purified shuffled AAV were then incubated withdifferent cell lines (in 6 cm dishes), together with also varyingamounts of helper Adenovirus type 5. Ideally, the Adenovirus would lysethe cells within three days, giving the AAV sufficient time toreplicate. The AAV amounts were adjusted to obtain minimal signals inWestern blot analyses of cell extracts. This helped to optimize thestringency of the library in each amplification round, by ensuring thata single viral genome was delivered to each cell, and subsequentlypackaged into the capsid expressed from its own genome.

In one set of experiments, the library was additionally subjected toIVIG pressure during amplification. Therefore, various volumes of thelibrary and IVIG (Gamimune®N 10%, Bayer, Elkhardt, Ind., USA) were mixedand incubated for 1 hour at 37° C., and then added to the cells. Afterovernight incubation, the cells were washed and super-infected withAdenovirus. The wash step was included to avoid helper virusinactivation by the IVIG. As before, AAV amplification was controlled byWestern blotting after each round, and only supernatants giving minimalexpression were used for subsequent infections. The increasing IVIGresistance of the library during consecutive passages allowed continuousescalation of the IVIG doses. All amplification experiments comprisedfive infection cycles (Adenovirus was heat inactivated between each andthen added fresh, to avoid uncontrolled amplification). Finally, viralDNA was purified from the supernatant (DNA Extractor Kit, Wako, Japan),and AAV cap genes were PCR amplified (DeepVent Polymerase), usingprimers 5′ GATCTGGTCAATGTGGATTTGGATG 3′ (SEQ ID NO:10; binding in AAV-2rep upstream of the Pac I site used for cap cloning) and 5′GACCGCAGCCTTTCGAATGTCCG 3′ (SEQ ID NO:11; binding downstream of the AscI site and polyadenylation signal). The resulting blunt-ended cap geneswere subcloned using the Zero Blunt TOPO PCR cloning kit for Sequencing(Invitrogen) and DNA was prepared from individual clones (96 per cellline/amplification round).

To assemble full-length cap sequences, T3 and T7 primers were used toobtain the 5′ and 3′ ends of each clone, and then individual primers(not shown) were designed to acquire the remaining sequence. Alignments(DNA and protein) with the eight parental cap genes were performed usingBLAST and VectorNTI 10/AlignX software (Invitrogen).

D. AAV Protein Analyses

Western blot and immunofluorescence analyses were carried out asreported (Grimm, D. et al., Blood, 102:2412-2419 (2003)) using themonoclonal B1 antibody for detection of immobilized AAV capsid proteins,useful because its eight amino acid epitope is largely conserved acrossknown AAV serotypes.

Example 2 In Vitro Transduction with Recombinant AAV-DJ Vectors

A. Helper Plasmid Cloning and Vector Particle Production

Helper plasmids expressing wildtype AAV-2, -8 or -9 cap together withAAV-2 rep genes, as well as AAV-2-based vector plasmids expressing thehFIX gene from a liver-specific or the EF1α promoter, were previouslydescribed (Nakai. H. et al., J. Virol., 79:214-224 (2005)); Gao, G. etal., J. Virol., 78:6381-6388 (2004)). Two self-complementary vectorplasmids expressing either the gfp gene from a CMV promoter, or the hAATgene from an RSV (Rous Sarcoma Virus) promoter, were prepared usingconventional techniques.

For cloning of helper plasmids expressing shuffled cap genes, the entireAAV-8 cap gene was removed from the AAV-8 helper construct by cuttingwith Swa I and Pme I (both create blunt ends; Swa I cuts 9 nt upstreamof the VP1 start codon, Pme I cuts 53 nt downstream of thepolyadenylation signal). The novel cap genes were amplified from therespective TOPO constructs (see above) via PCR, using the forward primer5′ AAAT CAGGT 3′ (SEQ ID NO:12; the underlined nt restored the Swa Isite to maintain correct reading frames) directly attached at the 3′ endto the first 25 nt of each cap gene, which for AAV-DJ was:ATGGCTGCCGATGGTTATCTTCCAG (SEQ ID NO:13; identical in AAV-2, -8 and -9).The reverse primer was 5′ AAACAATTCGCCCTTCGCAGAGACCAAAGTTCAACTGAAACGAATCAACCGG TTTATT GATTAACAGGCAA 3′(SEQ ID NO:14; nt restoring the Pme I site are underlined, thepolyadenylation signal is shown in bold) directly attached at the 3′ endto the last (3′) 23 nt of the shuffled capsid genes, which for AAV-DJwas: TTACAGATTACGGGTGAGGTAAC, 3′-5′ orientation, SEQ ID NO:15). PCRswere performed using DeepVent DNA Polymerase (NEB), creating blunt endsallowing straight-forward insertion into the linearized AAV-8 helperplasmid. Insert junctions and correct orientation were confirmed via DNAsequencing (Biotech Core). Vector production and particle titration (dotblot) were performed as previously described (Nakai, H. et al., J.Virol., 79:214-224 (2005)). Yields for all vectors including AAV-DJ andthe HBO mutants typically exceeded 6×10¹³ total physical particles per50×T225 flasks (2×10⁹ cells).

B. In Vitro Transduction

All transformed cell lines were maintained in DMEM (Gibco) containing10% fetal calf serum, 2 mM L-glutamine and 50 IU/ml of each penicillinand streptomycin at 37° C. in 5% CO₂. Fresh primary human hepatocytes(in 6-well plates without Matrigel) were obtained from Admet (Durham,N.C., USA) and maintained in Hepatocyte Basal Medium (Cambrex,Walkersville, Md., USA) with recommended supplements. Titration ofgfp-expressing recombinant AAV particles was performed in 96-wellplates, following normalization of each virus stock to 2×10⁹particles/mL. For in vitro neutralization studies, 50 μL per vectorpreparation were incubated with serial 10-fold dilutions of two batchesof IVIG (designated IVIG1 and IVIG2) or mouse sera (following a 1 hourheat inactivation at 56° C.) for 1 hour at 37° C. prior to titration oncells. Titers of neutralizing antibodies were calculated as reported(Grimm, D. et al., Blood, 102:2412-2419 (2003)).

Example 3 In Vivo Studies

Recombinant AAVs with either the AAV-DJ, AAV-2, AAV-8, or AAV-9 capsidswere produced by triple transfecting cells with a plasmid encoding thehuman factor IX (FIX) gene under the control of an adjacentliver-specific promoter and flanked by AAV inverted terminal repeats(ITRs), a plasmid encoding adenoviral helper genes, and a plasmidencoding the AAV-2 rep gene and either the AAV-DJ, AAV-2, AAV-8, orAAV-9 capsid protein. The liver-specific promoter that was used was ahuman alpha-1-antitrypsin (hAAT) promoter fused with an apolipoprotein E(ApoE) enhancer and an HCR (hepatic locus control region). The AAV ITRsthat were used were derived from AAV-2 and are identical to each other.

Doses of 5×10¹⁰, 2×10¹¹, and 1×10¹² (low, medium, and high,respectively) recombinant viral particles were injected into mice viatail vein infusions with a volume of 300 microliters infused over aperiod of about 10 seconds. The mice were bled at 1, 3, and 8 days afterthe infusions. The FIX protein plasma levels were quantified by ELISA,and the results are shown in FIG. 4.

Example 4 In Vivo Studies

A. Expression Studies in Mice

Wildtype female C57/BL6 mice (6 to 8 weeks old, 20 to 25 grams) werepurchased from Jackson Laboratory (Bar Harbor, Me., USA). RecombinantAAV expressing the hFIX or hAAT genes were administered in 300 μL 1×PBSvia tail vein infusion. Blood was collected at the indicated timepointsvia retroorbital bleeding, and plasma hFIX or hAAT levels weredetermined via ELISA as previously described (Nakai, H. et al., J.Virol., 79:214-224 (2005); Grimm, D. et al., Nature, 441:537-541(2006)). Results for the hAAT-expressing vectors are shown in FIG. 5.

Genomic DNA was extracted from mouse tissues and analyzed via Southernblotting, using hFIX- or hAAT-specific probes, as previously reported(Nakai, H. et al., J. Virol., 76:11343-11349 (2002)).

B. Immunologic In Vivo Assays

For passive immunization studies, mice (n=4 per group) were injectedintravenously (tail vein) with 40 μL (low dose) or 200 μL (high dose) ofIVIG (100 mg/mL) diluted in 1×PBS to a total volume of 300 μL, and 24hours later infused (tail vein) with 2×10¹¹ recombinant hFIX-expressingAAV. Plasma hFIX levels per virus and timepoint are shown in FIGS. 6A-6Bas percent of corresponding levels in control mice (PBS instead ofIVIG).

For cross-neutralization studies, mice were immunized against individualAAV serotypes by peripheral infusion of 1×10¹¹ recombinanthAAT-expressing particles. Three weeks later, mouse serum was collectedfor in vitro neutralization assays, before the mice were re-infused with1×10¹¹ (5×10¹¹ for AAV-2) recombinant hFIX-expressing AAV. Sera wastaken from the mice at the time of re-injection (H), as well as from aparallel group of mice injected with a lower dose (L) of 2×10¹⁰particles. Neutralizing antibody titers (NAb) against the wildtype AAVsor AAV-DJ were determined. Results are shown in FIGS. 6C-6D.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

What is claimed is:
 1. A method of generating a library of recombinantAAV plasmids, comprising: isolating AAV capsid nucleotide sequences fromtwo or more serotypes of AAV; digesting the AAV capsid nucleotidesequences into fragments; reassembling the fragments using PCR to formre-assembled PCR products; and cloning the re-assembled PCR productsinto plasmids to generate a library of recombinant AAV plasmids.
 2. Themethod of claim 1, wherein the reassembling the fragments using PCR isusing primer-less PCR.
 3. The method of claim 1, wherein isolatingincludes isolating AAV capsid nucleotide sequences from human AAVserotypes and non-human AAV serotypes.
 4. The method of claim 3, whereinisolating includes isolating AAV capsid nucleotide sequences selectedfrom the group consisting of AAV-2, AAV-8, and AAV-9.
 5. The method ofclaim 1, further comprising, after said cloning, transfecting cells withthe plasmids to produce a viral library.
 6. The method of claim 5,wherein said transfecting comprises transfecting 293 kidney cells with ahelper Adenovirus.
 7. The method of claim 5, further comprising, aftersaid transfecting, passaging the viral library in a selected cell typein the presence of a stringent condition, and selecting AAV capsids thatsurvive said passaging.
 8. The method of claim 7, wherein the stringentcondition comprises the presence of human immune globulin.
 9. Arecombinant AAV library prepared according to the method of claim 1.